Project Summary Enteric microbiota interact with STING and Myd88 pathways to reinforce healthy mucus secretion and innate immune activation in the intestine. Mucus dysregulation precipitates microbe-induced inflammation, a driver of GI pathologies, but the mechanisms underpinning this process are poorly understood. If we better understood the relationship between host mucus and bacterial physiology at the molecular level, we could design therapies to alleviate inflammation that arises from mucus dysregulation. The bacterial second messenger c-di-GMP (cdG), which promotes bacterial aggregation in response to environmental cues, stimulates vertebrate innate immune STING signaling. The larval zebrafish, Danio rerio, is a powerful model for exploring aspects of host-microbe interactions conserved in humans. My recent studies indicate a zebrafish mutualist Aeromonas veronii ZOR0001(A01), which secretes beneficial factors conserved in human-associated microbiota, increases cdG levels in response to mucus, leading to upregulation of a mucus-binding adhesin, and A01 aggregation in the mucus-rich mid gut of the larval zebrafish intestine. Various bacteria increase cdG when exposed to mucus, indicating this concept is a conserved adaptive strategy in bacteria. We hypothesize that mucus-mediated A01 aggregation promotes healthy mucus secretion, setting a proper inflammatory tone to prevent excess inflammation. Consistent with this hypothesis I observe highly motile A01 in a zebrafish genetic background with decreased mucus-secreting cells. Furthermore, I found that treating a zebrafish mutant prone to spontaneous intestinal inflammation with a A01 hyper-aggregating mutant that produces high levels of cdG can alleviate intestinal neutrophil influx. Therefore, we propose to characterize the molecular basis of A01 cdG signaling in the host and determine if normal cdG-STING signaling contributes to intestinal homeostasis. Specific Aims: (1) determine the genetic basis by which host mucus prompts A01 cdG synthesis, and (2) investigate how STING sensing of A01-produced cdG modulates host inflammation and intestinal mucus homeostasis in vivo. Research Design: Using my A01 cdG reporter strain, I will first screen for A01 genes involved in mucus-mediated cdG synthesis in vitro and compare intestinal cdG signaling and distribution patterns of these mutants to wild type A01 in the zebrafish intestine. To understand the role of cdG regulation in mutualism and host intestinal health, A01 mutants deficient in mucus-sensing will be monoassociated into transgenic larval zebrafish that allow quantification of intestinal mucus-secreting cells and inflammation responses. I will also generate sting deficient zebrafish incapable of sensing cdG and compare, through phenotypic characterization and single cell RNAseq, how they respond to A01 and bacterial products such as cdG and LPS relative to wild type and myd88 zebrafish. This research will improve our understanding of biologically and medically important processes that underpin host-microbe interactions. Because rates of inflammatory GI disorders are on the rise, the long-term goal of our work is to translate our findings into strategies for promoting intestinal health in humans.